Date of Graduation

Document Type

Program Affiliation

Degree Name

Advisor/Committee Chair

Committee Member

Rodney E. Kellems, Ph.D.

Committee Member

Cheng Chi Lee, Ph.D.

Committee Member

Pierre D. McCrea, Ph.D.

Committee Member

Jianping Jin, Ph.D.

Committee Member

Rachel K. Miller, Ph.D.

Abstract

The connecting tubule interconnects the nephron and collecting duct, which arise from kidney mesenchyme and the ureteric bud, respectively, to generate the functional tubular networks. The collecting duct is comprised of principal cells and intercalated cells, which bear different molecular signatures and regulate sodium/water and acid/base balance, respectively. The progenitor cells of the connecting tubule and the collecting duct remain virtually unknown.

We generated two Aqp2 lineage tracing mouse models. In these models, Aqp2Cre transgene drives Cre expression by the Aqp2 promoter to exclusively either inactivate histone H3 K79 methyltransferase Dot1l (Dot1lf/f Aqp2Cre) or activate RFP in Aqp2 lineage cells during development (Aqp2Cre RFP). H3 K79methylation and RFP were used as the tracing markers. Kidney sections were examined by immunofluorescence staining combined with epifluorescence and confocal microscopy.

Analyses of Dot1lf/f Aqp2Cre revealed that Dot1l ablation abolished H3 K79 methylation, which occurs in both principal and intercalated cells. These results suggest that Aqp2+ progenitor cells give rise to principal cells and intercalated cells in the absence of Dot1l function. With Aqp2Cre RFP mice, we not only confirmed that derivation of intercalated cells from Aqp2+ progenitor cells is not an artifact of Dot1l deletion, but also identified the origin and molecular identities of connecting tubules. Aqp2+ progenitors contribute to renal tubular interconnection by differentiating into various types of transitional cells in the connecting tubule to form three molecularly distinct segments: RFP+Aqp2+NCC-, RFP+Aqp2-NCC-, and RFP+Aqp2-NCC+. RFP+ indicates progenitors of Aqp2+ origin. Aqp2- represents the loss of the original Aqp2+ progenitors. NCC+is the signature of distal convoluted tubule, the last segment ofnephron linking to the connecting tubule.

In summary, our study 1) highlights the molecular identity and the origin of novel and distinct connecting tubule segments; and 2) reveals Aqp2+ progenitors as the origin of various cell types of connecting tubule as well as collecting duct. Therefore, our study demonstrates novel functions of Aqp2+progenitors in the origin of collectingduct and connecting tubule formation. The discovery of the Aqp2+ progenitor cells may facilitate their further molecular and functional characterization, which is critical for regenerative medicine.